Abstract Streptococcus mitis is a highly abundant Gram-positive human oral commensal and opportunistic pathogen causing bacteremia and infective endocarditis. Despite its relevance to human health, S. mitis is critically understudied, and very little is known about its fundamental cell biology. We have identified several novel aspects of S. mitis physiology which likely have significant ramifications for its interactions with antimicrobials and with the host: 1. S. mitis rapidly evolves resistance to the cationic antibiotic daptomycin (DAP) via complete removal of the anionic phospholipids phosphatidylglycerol (PG) and cardiolipin (CL) from the membrane. Because PG and CL have important housekeeping and stress response functions in bacteria, our results suggest that S. mitis possesses novel physiological mechanisms that allow them to survive and replicate without these lipids. 2. S. mitis synthesizes two structurally distinct lipoteichoic acid (LTA) polymers and switches between them in response to growth environment. This is the first evidence for LTA switching in a Gram-positive pathogen and has ramifications for the interactions of S. mitis with the host. 3. S. mitis synthesizes phosphatidylcholine (PC), a zwitterionic phospholipid with a positively charged head group, by scavenging PC breakdown products from the host. We show that PC is non-essential in vitro for S. mitis, suggesting that S. mitis could regulate PC levels in the membrane as a means to modulate surface charge. The long-term, overarching hypothesis of this project is that the ability of S. mitis to drastically alter lipid compositions in the cellular envelope is critical for its habitat flexibility in the host and its survival in response to antimicrobial stress. The Aims are: Aim 1. Comprehensive 'omics analysis of S. mitis lacking major anionic phospholipids. Aim 2. LTA and phosphatidylcholine biosynthesis in S. mitis. Aim 3. Roles of lipids in S. mitis- host and S. mitis-antimicrobial interactions.